Abstract: Cytometry Facility (CF) Cytometry is a mainstay of cancer cell biology and immunology research and, as a single-cell multiparameter analytical tool, provides a key to deciphering cellular heterogeneity. As a preparative method, flow cytometric sorting is rapidly becoming integrated with genomic and transcriptomic studies, permitting molecular analysis of well-characterized single sorted cells, or phenotypically homogeneous cell populations. Both analytical and preparative techniques are particularly well-suited to rare event problems, with cancer applications ranging from detection of circulating and disseminated cancer cells, monitoring minimal residual disease, and defining the role of cancer cell subsets within heterogeneous tumors. The UPCI Cytometry Facility (CF), a UPCI- managed shared resource and located in the Hillman Cancer Center Research Pavilion, provided services to more than 100 users over the past funding period. The CF has 5 specific aims, to: 1) Train users to operate the Facility?s four analytical flow cytometers; 2) Perform high-speed biocontained fluorescence activated cell sorting for investigators; 3) Perform rigorous QA/QC to ensure that the instruments in the facility are properly calibrated on a daily basis and advising users concerning the proper settings for their experiments; 4) Advise investigators on experimental design and data analysis and assisting in performance of complex multi- parameter experiments; and 5) Archive all data produced by the facility. The current instrumentation at the CF includes a Beckman-Coulter (BC) MoFlo Astrios high speed cell sorter, equipped with a BSL Class II containment cabinet, a Becton Dickinson (BD) LSR2 Fortessa SORP 4-laser analyzer, a BC 3-laser analyzer, 2 BD Accuri 2-laser analyzers, and a ThermoFisher/Cellomics VTI100 high throughput imaging system. CF upgrades during the past funding period were extensive, including the Astrios sorter (SIG RR032768-01), and the Fortessa, Gallios and Accuri analyzers (all purchased with UPCI funds). CF informatics upgrades include a gigabit link to a new high capacity file server maintained by UPMC, and iLAB, a new web-based scheduling/billing system. The usage of the CF has remained high, with cell sorting regularly exceeding 50% of absolute capacity and analytical cytometry exceeding 50% of business-hour capacity. Future plans of the CF include a collaborative initiative to develop an interface between rare event sorting and imaging cytometry, providing high resolution confocal images of sorted cells for signaling studies (nuclear translocation, phosphorylation), and live-cell imaging of matrix experiments in which labeled cells are sorted in varying proportions directly into culture wells and outcomes are measured by microscopic imaging. During current project period members of all 10 UPCI Research Programs used the CF.